Antitrypanosomal Quinoline Alkaloids from the Roots of Waltheria

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Antitrypanosomal Quinoline Alkaloids from the Roots of Waltheria indica Sylvian Cretton,† Lise Breant,† Lucie Pourrez,† Chiara Ambuehl,† Laurence Marcourt,† Samad Nejad Ebrahimi,‡,§ Matthias Hamburger,‡ Remo Perozzo,† Soumana Karimou,# Marcel Kaiser,⊥,∥ Muriel Cuendet,† and Philippe Christen*,† †

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland ‡ Division of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland § Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran # Plantasav, B.P. 10308 Niamey, Niger ⊥ Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland ∥ University of Basel, Petersplatz 1, 4003 Basel, Switzerland S Supporting Information *

ABSTRACT: Chemical investigation of the dichloromethane root extract of Waltheria indica led to the isolation and characterization of 10 quinoline alkaloids, namely, 8deoxoantidesmone (1), waltheriones E−L (2−9), and antidesmone (10). Among these, compounds 2−9 have not yet been described in the literature. Their chemical structures were established by means of spectroscopic data interpretation including 1H and 13C NMR, HSQC, HMBC, COSY, and NOESY experiments and UV, IR, and HRESIMS. The absolute configurations of the compounds were established by comparison of experimental and TDDFT-calculated ECD spectra. In addition, the isolated constituents were evaluated for their in vitro antitrypanosomal activity. Compounds 4, 5, and 8 showed potent and selective growth inhibition toward Trypanosoma cruzi with IC50 values between 0.02 and 0.04 μM. Cytotoxicity for mouse skeletal L-6 cells was also determined for these compounds. and suramin for the first stage and melarsoprol, eflornithine, and the nifurtimox−eflornithine combination for the second stage of the disease.5 Another parasite of the Trypanosoma genus, Trypanosoma brucei brucei (T. b. brucei), is pathogenic to wild and domestic animals. In cattle, the disease is called “Nagana” and is a major obstacle to the economic development of affected rural areas.4 Waltheria indica L. (Malvaceae) is a short-lived shrub that can reach up to 2 m in height and is widespread in subtropical and tropical regions of the world. In traditional medicine, the roots, aerial parts, and whole plant either are taken as a decoction, infusion, or macerate or are chewed or directly applied on wounds. Many ailments such as skin ulcers, rheumatism, diarrhea, hemorrhoids, asthma, or tooth infections are treated with this plant.6 In Burkina Faso, the roots and aerials parts are used against malaria,7 and in Niger and Nigeria,

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merican trypanosomiasis (AT), also known as Chagas disease, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi (T. cruzi). About 7 to 8 million people are estimated to be infected worldwide, mostly in Latin America, where Chagas disease is endemic.1 AT is characterized essentially by two clinical forms, an acute stage and a chronic stage. To date, benznidazole and nifurtimox are the only two available chemotherapeutic agents. These compounds are effective for the acute phase of the disease. In contrast, during the chronic phase their utility is limited due to low efficacy.2,3 In addition, both drugs produce severe side effects occurring in up to 40% of treated patients.1 Human African trypanosomiasis (HAT), also known as sleeping sickness, is a fatal disease caused by Trypanosoma brucei rhodesiense (T. b. rhodesiense) and Trypanosoma brucei gambiense (T. b. gambiense). It threatens millions of people in 36 countries in sub-Saharan Africa4 and progresses in two stages. In the first stage, the trypanosomes are located mainly in the hemolymphatic system. In the second stage, the parasites overcome the blood−brain barrier and invade the brain. Few drugs are available to treat the disease and include pentamidine © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 19, 2014

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C-10, respectively), a methine at δC 32.3 (C-5), 10 methylenes at δC 33.7, 33.1, 31.0, 30.8, 30.5, 29.0, 27.6, 25.8, 23.7, and 18.0 (C-11, C-16, C-12 to C-15, C-8, C-6, C-17, and C-7, respectively), a methoxy at δC 60.3, and two methyls at δC 14.4 and 13.5 (C-18 and CH3-2, respectively). The HMBC correlations from the methoxy to C-3, from the methyl CH3-2 to C-2 and C-3, from H-5 to C-6, C-7, C-9, C-10, and C-11, and from CH3-18 to C-17 and C-16 as well as the COSY correlations from H-5 (δH 2.91) to H2-6 (δH 1.91, 1.54) and H2-11 (δH 1.75, 1.26) and from H2-7 (δH 1.82, 1.77) to H2-6 and H2-8 (δH 2.62) led to the conclusion that 1 is 3-methoxy-2methyl-5-octyl-5,6,7,8-tetrahydroquinolin-4(1H)-one (8-deoxoantidesmone, Figure 1).

traditional healers give the whole plant to cattle as a tonic, suggesting a possible activity against “Nagana”.8 Although W. indica is commonly used in traditional medicine, very few phytochemical studies have been performed on this plant. Flavonoids and four cyclopeptide alkaloids named adouetins X, Y, Y1, and Z were isolated and identified from the whole plant or aerial parts.9−13 Dichloromethane extracts of the aerial parts and roots of W. indica were prepared and screened with the aim of discovering new natural products with antitrypanosomal activity. This contribution describes the isolation and characterization of 10 quinoline alkaloids from the roots of W. indica. Among these, eight compounds (2−9) are new natural products. The antitrypanosomal activity of the isolated compounds was then determined.

Figure 1. Key COSY (bold lines) and HMBC (arrows) correlations of 1.

This molecule (1) has already been described from plants classified in the tribe Antidesmeae (Euphorbiaceae) by Buske et al.14 However, this is the first time that 8-deoxoantidesmone has been isolated and characterized using NMR spectroscopy. The absolute configuration at C-5 was established by comparison of experimental and calculated ECD spectra. The experimental ECD spectrum exhibited positive and negative Cotton effects (CEs) at 285 and 261 nm, respectively. The corresponding absorption bands could be attributed to π → π* electronic transition from the bonding to antibonding molecular configuration. The calculated ECD spectrum for the (5R)-stereoisomer showed an excellent fit with the experimental data, with positive and negative CEs around 295 and 260 nm, respectively (Figure 2). Thus, the absolute configuration of compound 1 was established as (5R)-8deoxoantidesmone. The HRESIMS of compound 2 exhibited an [M + H]+ ion at m/z 336.2534 (calcd for C20H34NO3, 336.2539), corresponding to the molecular formula C20H33NO3. In comparison with 1, an extra methoxy group (+30 amu) was confirmed by NMR spectroscopy, where an additional methine at δH 4.23 (1H, dd, J = 10.5, 5.9 Hz, H-8) and a methoxy group at δH 3.43 (3H, s, OCH3-8) were detected instead of the methylene on C-8. The HMBC correlations between OCH3-8 and C-8 and between H8 and C-9 led to the unequivocal location of the second methoxy group at C-8. It is worth noting that an isomer of 2, bearing a methoxy group in C-7 instead of C-8 and named chamaedrone, was isolated from Melochia chamaedrys (Malvaceae).15 In addition, in the same study, two enantiomers of 2, (5S,8R) and (5S,8S), were produced semisynthetically from antidesmone. Unfortunately, neither NMR spectra nor optical rotation data were provided for these two enantiomers, precluding the determination of the relative configuration at C-8. To establish the absolute configuration, the ECD spectrum of 2 was measured and compared with calculated spectra of the two possible stereoisomers, (5S,8S) and (5S,8R). The

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RESULTS AND DISCUSSION The powdered, air-dried roots of W. indica were extracted with dichloromethane at room temperature. After filtration and evaporation under reduced pressure, the residue was separated by medium-pressure liquid chromatography (MPLC) into 320 fractions by RP C18 silica gel column chromatography. Compounds 1−3 were readily isolated. Further purifications of the fractions were carried out by repeated column chromatography and afforded seven additional compounds, 4−10. High-resolution electrospray ionization mass spectrometry (HRESIMS) measurement of all compounds suggested the presence of a nitrogen atom in the molecular formula, indicating the putative presence of alkaloids. NMR data (Tables 1 and 2) and 2D NMR experiments suggested a quinoline skeleton for this series of compounds. HRESIMS of compound 1 showed a pseudomolecular ion peak at m/z 306.2428 [M + H]+ (calcd for C19H32NO2, 306.2433), indicating a molecular formula of C19H31NO2. The 13 C NMR and HSQC spectra revealed the presence of a carbonyl group at δC 174.0 (C-4), four quaternary aromatic carbons at δC 145.6, 144.8, 140.4, and 129.4 (C-3, C-9, C-2, and B

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3. Indeed, in the 1H and 13C NMR spectra two aromatic protons at δH 6.93 (1H, d, J = 8.0 Hz, H-6) and 7.03 (1H, d, J = 8.0 Hz, H-7) were observed instead of two methylene protons in 2 and two quaternary aromatic carbons at δC 136.7 (C-5) and 147.9 (C-8) instead of the two methine carbons in 2 (Table 2). Compound 3 was thus identified as 3,8-dimethoxy2-methyl-5-octylquinolin-4(1H)-one. Compound 4 (waltherione G) showed an [M + H]+ ion at m/z 370.2378 (calcd for C23H32NO3, 370.2382), corresponding to the molecular formula C23H31NO3. When compared with 1, the 1H NMR spectrum of 4 displayed five aromatic protons at δH 7.23 (2H, t, J = 7.6 Hz, H-18, 20), 7.16 (2H, d, J = 7.6 Hz, H-17, 21), and 7.13 (1H, t, J = 7.6 Hz, H-19) and an additional methoxy group at δH 3.99, whereas the CH3-18 signal of 1 was missing. The same conclusion was made from the 13C NMR spectrum of 4, where eight methylene signals were detected between δC 18.2 and 36.9 instead of 10 for compound 1. The long-range heteronuclear correlations between H-17/H-21 and C-15 (δC 36.9) and between H2-14 (δH 1.64) and C-16 (δH 144.0) could be used to position unambiguously the phenyl group at the terminal position of the n-pentyl side chain (Figure 4). The additional methoxy group was positioned on the

Figure 2. Experimental ECD spectra of 1 and 6 and comparison with the calculated spectrum for the (5R)-stereoisomer. The calculation was achieved with TDDFT at the B3LYP/6-31** level in MeOH.

experimental spectrum showed two positive CEs at 226 and 265 nm and one negative CE at 285 nm (Figure 3). These

Figure 4. Key NOESY (dashed arrows) and HMBC (plain arrows) correlations of 4.

nitrogen atom, based on the NOE correlations observed between this methyl and both CH3-2 (δH 2.46) and H-8 (δH 2.74 and 3.01). The ECD spectrum of compound 4 showed two positive CEs at 232 and 265 nm and one negative CE at 295 nm. As compared to compound 1, the spectrum was opposite in sign. Given that 4 has one chiral center, the opposite configuration at C-5 was expected. The calculated ECD spectrum of the (5S)stereoisomer matched with the experimental data (Figure 5) and confirmed the absolute configuration at C-5 as S. Therefore, the structure of 4 could be assigned as 1,3dimethoxy-2-methyl-5-(5-phenylpentyl)-5,6,7,8-tetrahydroquinolin-4(1H)-one. The alkaloid 5 (waltherione H) was found to be similar to 4 except that an additional methoxy group was present as indicated by the molecular formula, C24H34NO4, deduced from the ion m/z 400.2483 [M + H]+ instead of C23H32NO3 for 4. The third methoxy group at δH 3.48 was positioned at C-8 (δC 71.9) based on the HMBC correlation. The absolute configurations of C-5 and C-8 were assigned as (5S,8R) by comparison of the experimental and calculated ECD spectra (Figure 3). Alkaloid 5 was thus identified as 1,3,8-trimethoxy-2methyl-5-(5-phenylpentyl)-5,6,7,8-tetrahydroquinolin-4(1H)one. Compounds 4 and 5 are closely related to melovilone (3,7,8-trimethoxy-2-methyl-5-(5-phenylpentyl)quinolin-4(1H)one), an alkaloid isolated previously from Melochia tomentosa.16

Figure 3. Experimental ECD spectra of 2, 5, and 8 and comparison with calculated spectra for the two possible [(5S,8R) and (5S,8S)] stereoisomers.

features in the UV and ECD spectra are mainly due to π → π* transitions of the quinoline chromophore. The calculated spectrum for the (5S,8R)-stereoisomer showed two positive CEs at 225 and 260 nm together with a negative CE at 280 nm. In contrast, the calculated ECD data of the (5S,8S)-stereoisomer showed a CE of an opposite sign at 225 nm when compared to the experimental spectrum. Hence, the good match between the experimental spectrum and the calculated data for the (5S,8R)-stereoisomer led to the conclusion that the absolute configuration of 2 is (5S,8R). Compound 2 was identified as 3,8-dimethoxy-2-methyl-5-octyl-5,6,7,8-tetrahydroquinolin-4(1H)-one and named waltherione E. Compound 3 (waltherione F) exhibited a pseudomolecular ion peak at m/z 332.2220 [M + H]+ (calcd for C20H30NO3, 332.2226), indicating a molecular formula of C20H29NO3 and implying two additional degrees of unsaturation than 2. The NMR data confirmed that instead of a cyclohexene ring fused to a 4-pyridinone moiety as for 2, a benzene ring was found in C

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ECD spectrum of 6 (Figure 2) was identical to that of compound 1, and the configuration at C-5 was determined as (5R). Alkaloid 6 was identified as 2-(hydroxymethyl)-1,3dimethoxy-5-octyl-5,6,7,8-tetrahydroquinolin-4(1H)-one and named waltherione I. The latter is similar to vanessine, a tetrahydroquinoline alkaloid isolated previously from Waltheria douradinha.17 However, the absolute configuration (5R) of 6 determined by ECD was found to be opposite of that suggested for vanessine (5S). Compound 7 was found to share the same molecular formula, C20H34NO3, as 2, suggesting that they are isomeric compounds. The 1H and HSQC spectra of 7 indicated the presence of 10 methylene groups between δC 17.5 and 33.4 (one more than 2). Like for compounds 4 and 5, the methoxy group at δH 3.99 (3H, s, OCH3-1) was positioned on the nitrogen atom, as it gave no HMBC cross-peak and showed a NOE with the methyl at δH 2.47 (3H, s, CH3-2). All these data led to the structure for 7 as 1,3-dimethoxy-2-methyl-5-octyl5,6,7,8-tetrahydroquinolin-4(1H)-one (waltherione J). The absolute configuration at C-5 was assigned as (5S) (Figure 5), identical to isomer 2. The HRESIMS of compound 8 led to the molecular formula C21H35NO4. In comparison to 7, the 1H spectrum displayed an additional methoxy group at δH 3.48 (3H, s, OCH3-8). A longrange correlation between C-8 and OCH3-8 was used to locate the latter without ambiguity. Compound 8 (waltherione K) was identified as 3,8-trimethoxy-2-methyl-5-octyl-5,6,7,8-tetrahydroquinolin-4(1H)-one. The ECD spectrum of alkaloid 8 was similar to that of 2, with two positive CEs at 225 and 250 nm

Figure 5. Experimental ECD spectra of 4 and 7 and comparison with the calculated spectrum for the (5S)-stereoisomer.

Compound 6 showed a pseudomolecular ion peak at m/z 352.2483 [M + H]+ (calcd for C20H34NO4, 352.2488), indicating a molecular formula of C20H33NO4. The structure of 6 was close to that of 1 except that the methyl on C-2 was replaced by a hydroxymethyl at δH 4.76 and that an additional methoxy group was at δH 4.12 attached to the nitrogen atom. The NOE observed between CH2OH and both OCH3-1 and OCH3-3 was used to position unambiguously the hydroxymethyl on C-2 and the methoxy on the nitrogen atom. The

Table 1. 1H NMR Data of Compounds 1−9 (500 MHz, in CD3OD except for 2 in CDCl3) δH (J in Hz) position

1

2

5

2.91, m

2.88, m

6

1.91, brd (13.4) 1.54, m

7

1.82, m 1.77, m 2.62, m

1.92, m 1.58, dtd (13.4, 7.1, 3.5) 2.07, dt (9.7, 3.5) 1.70, m 4.23, dd (10.5, 5.9)

8 11 12 13 14 15 16 17 18 19 20 21 OCH3-1 CH3-2 CH2OH-2 OCH3-3 OCH3-4 OCH3-8

1.75, 1.26, 1.41, 1.34, 1.34, 1.34, 1.34, 1.32, 0.90,

m m m m m m m m t (6.5)

2.30, s 3.76, s

1.78, 1.29, 1.38, 1.24, 1.24, 1.24, 1.24, 1.22, 0.84,

m m m m m m m m t (6.8)

2.30, s

3

4

5

2.95, m

2.92, m

6.93, d (8.0)

1.86, m 1.50, m

1.87, m 1.70, m

7.03, d (8.0)

1.80, m

2.25, d (12.0) 1.35, m 4.67, s

3.27, m 1.59, 1.32, 1.30, 1.30, 1.30, 1.30, 0.89,

m m m m m m t (6.2)

2.48, s

3.79, s

3.78, s

3.43, s

4.00, s

3.01, 2.74, 1.76, 1.25, 1.44, 1.39, 1.64, 2.60,

m m m m m m m t (7.7)

1.99, 1.35, 1.33, 1.33, 1.61, 2.58,

m m m m m t (7.7)

7.16, d (7.6) 7.23, t (7.6)

7.15, d (7.2) 7.22, t (7.6)

7.13, 7.23, 7.16, 3.99, 2.46,

7.12, 7.22, 7.15, 4.16, 2.49,

t (7.6) t (7.6) d (7.6) s s

3.77, s

t (7.2) t (7.6) d (7.2) s s

3.77, s 3.48, s D

6

7

8

9

2.98, dt (10.6, 3.1) 1.92, m 1.55, m

2.95, m

2.91, m

3.03, m

1.91, m 1.52, m

1.87, m 1.68, m

1.95, m 1.52, m

1.84, m

1.80, m

1.87, m

1.90, m

2.77, d (10.4)

3.02, m 2.75, m 1.72, m 1.25, m 1.41, m 1.32, m 1.32, m 1.32, m 1.32, m 1.32, m 0.90, t (6.5)

4.67, s

2.63, t (7.8)

1.98, m 1.39, m 1.30, m 1.29, m 1.29, m 1.29, m 1.29, m 1.29, m 0.89, t (6.6)

1.69, m

1.72, 1.29, 1.41, 1.32, 1.32, 1.32, 1.32, 1.32, 0.90,

m m m m m m m m t (6.7)

1.30, 1.30, 1.40, 2.63,

m m m t (7.7)

7.17, d (7.3) 7.24, t (7.6) 7.14, t (7.3) 7.24, t (7.6) 7.17, d (7.3)

4.12, s 4.76, s 3.85, s

3.99, s 2.47, s

4.16, s 2.49, s

3.77, s

3.78, s

2.47, s 3.83, s 3.99, s

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Table 2. 13C NMR Data of Compounds 1−9 (125 MHz, in CD3OD except for 2 in CDCl3) δC, type position 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 OCH3-1 CH3-2 CH2OH-2 OCH3-3 OCH3-4 OCH3-8 a

1 140.4, 145.6, 174.0, 32.3, 25.8, 18.0, 27.6, 144.8, 129.4, 33.7, 29.0, 30.8, 31.0, 30.5, 33.1, 23.7, 14.4,

2 C C C CH CH2 CH2 CH2 C C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

13.5, CH3 60.3, CH3

137.5, 144.8, 172.9, 31.2, 22.8, 22.4, 75.3, 140.7, 129.1, 32.6, 28.4, 29.4, 29.9, 29.8, 32.0, 22.8, 14.2,

3 C C C CH CH2 CH2 CH C C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

14.4, CH3

142.8, 143.2, 175.9, 136.7, 125.4, 110.6, 147.9, 132.4, 125.0, 36.4, 33.7, 30.8, 30.9, 30.6, 33.1, 23.7, 14.4,

4 C C C C CH CH C C C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

14.1, CH3

59.6, CH3

60.2, CH3

56.1, CH3

56.6, CH3

141.0, 145.4, 172.6, 32.7, 25.7, 18.2, 24.9, 145.1, 129.7, 33.5, 28.8, 30.5, 32.8, 36.9, 144.0, 129.2, 129.2, 126.6, 129.2, 129.2, 66.1, 10.4,

5 C C C CH CH2 CH2 CH2 C C CH2 CH2 CH2 CH2 CH2 C CH CH CH CH CH CH3 CH3

6

140.5, C 144.7, C a

146.2, C a

33.5, CH 22.6, CH2 a

32.8, CH a a

71.9, CH

24.9, CH2

140.9, 145.3, 172.6, 32.5, 25.2, 17.5, 24.2,

a

a

a

a

a

a

34.9, 27.4, 30.2, 32.5, 36.6, 142.5, 129.0, 129.0, 126.3, 129.0, 129.0, 67.7, 10.1,

60.3, CH3

7

a

CH2 CH2 CH2 CH2 CH2 C CH CH CH CH CH CH3 CH3

60.1, CH3 56.1, CH3

33.1, 28.2, 30.9, 30.8, 30.5, a 23.8, 14.4,

CH2 CH2 CH2 CH2 CH2 CH2 CH3

67.7, CH3 54.4, CH2 61.3, CH3

33.4, 28.8, 30.7, 30.8, 30.3, 32.9, 23.6, 14.3,

8 C C C CH CH2 CH2 CH2

9

140.5, C 145.4, C a

32.5, 24.6, 22.7, 72.3, 144.5,

CH CH2 CH2 CH C

a

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

35.1, 27.4, 30.4, 31.0, 30.7, 33.0, 23.7, 14.4,

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

66.2, CH3 10.2, CH3

67.3, CH3 14.4, CH3

60.1, CH3

60.1, CH3

141.2, 145.7, 152.0, 32.9, 24.8, 17.2, 36.9, 142.5, 127.8, 32.2, 30.9, 30.9, 30.0, 36.8, 142.4, 129.5, 129.5, 126.8, 129.5, 129.5,

C C C CH CH2 CH2 CH2 C C CH2 CH2 CH2 CH2 CH2 C CH CH CH CH CH

11.7, CH3 61.6, CH3 61.4, CH3

56.2, CH3

Signal too weak to be measured.

stereoisomer (Figure 7) showed an excellent fit with the experimental data. Thus, the absolute configuration at C-5 was

and a negative CE at 300 nm. It was concluded that the absolute configuration for 8 is (5S,8R) (Figure 3). The molecular formula of compound 9, C23H32NO3, determined from the ion m/z 370.2375 [M + H]+ was identical to that of 4. The NMR spectra of 4 and 9 exhibited strong similarities except that the carbonyl at δC 172.6 in 4 was replaced by a quaternary sp2 carbon at δC 152.0 and the methoxy group at δH 3.99 (OCH3-4) correlated with this carbon. The NOE correlations from OCH3-4 to OCH3-3 and H-5 and from OCH3-3 to CH3-2 were used to confirm this hypothesis (Figure 6).

Figure 7. Comparison of the experimental ECD spectrum of 9 with calculated data for the (5R)-stereoisomer.

Figure 6. Key NOESY correlations of 9.

However, the molecular formula of 9 indicated the presence of three oxygen atoms, but only two methoxy signals were observed in the 1H NMR spectrum. The only possibility for the location of the remaining oxygen was on the nitrogen atom. The ECD spectrum of 9 showed two positive CEs at 208 and 256 nm along with a negative CE at 222 nm. These were ascribed to a π → π* transition of the tetrahydroquinolin-1oxide moiety. The calculated ECD spectrum of the (5R)-

established as R. Compound 9 (waltherione L) was identified as 3,4-dimethoxy-2-methyl-5-(5-phenylpentyl)-5,6,7,8-tetrahydroquinolin-1-oxide. For compound 10, the molecular formula C19H29NO3, suggested by HRESIMS, and the NMR, UV, and IR data were consistent with literature values18 of antidesmone, a E

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Table 3. Antitrypanosomal and Cytotoxic Activity (IC50 in μMa) of Alkaloids 1−10 compound 1 2 3 4 5 6 7 8 9 10 suramin melarsoprol benznidazole podophyllotoxin

T. b. brucei >100 80.3 ± 31.0 ± 24.1 ± NDc >100 >100 >100 ND >100 0.31 ±

3.57 3.60 2.28

T. b. rhodesiense

T. cruzi

cytotoxicity

SIb for T. cruzi

32.6 38.5 38.6 30.4 35.9 87.9 53.3 45.8 55.7 14.3d

0.49 0.23 1.1 0.02 0.04 2.2 0.10 0.04 3.1 0.17d

0.75 0.91 25.7 0.64 0.26 22.1 0.19 0.07 41.1 184.3d

1.5 4.0 22.7 33.8 6.5 10.1 1.9 1.8 13.5 1084.3d

0.05e 0.01 2.9 0.02

Results are the means of two independent assays, except for T. b. brucei, where the assays were performed in triplicates and expressed ± SD. bSI (selectivity index) = IC50 cytotoxicity/IC50 antitrypanosomal activity. cND: not determined due to lack of product. dValues from the literature.20 e Values from the literature.20,21 a

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tetrahydroquinoline alkaloid isolated initially from Antidesma membranaceum (Euphorbiaceae).19 The dichloromethane extract of the roots of W. indica showed a strong in vitro inhibitory activity against T. cruzi (IC50 = 0.74 μg/mL) and T. b. brucei (2.3% survival at 20 μg/mL) and to a lesser extent against T. b. rhodesiense (IC50 = 17.4 μg/ mL). In addition, cytotoxicity toward mouse skeletal L-6 cells was considered as weak, with an IC50 = 24.0 μg/mL. On the basis of these encouraging results, the isolated alkaloids were tested in vitro for their antitrypanosomal and cytotoxic activities, and the data obtained are summarized in Table 3. Potent growth inhibition toward the amastigote form of T. cruzi (Tulahuen C4 strain) was measured for this series of alkaloids. Indeed, their IC50 values were lower than the reference drug benznidazole (IC50 = 2.9 μM) for all compounds except compound 9. The results showed that alkaloids 4 (IC50 = 0.02 μM) and 5 and 8 (IC50 = 0.04 μM each) displayed the most potent antichagasic activity. However, the cytotoxicity against L6 cells was high. From these results, it appears that a methoxy group bound to the nitrogen atom (compounds 4−8) increased lethality toward T. cruzi, suggesting that a nitrogen atom bearing a methoxy group is important for such activity, but further investigations must be performed to confirm this hypothesis. The configuration (5R) (compounds 1, 6, 9) seems to result in a decrease of activity. Moreover, the presence of an N-oxide function (alkaloid 9) may be detrimental for T. cruzi inhibitory activity. A comparison between the IC50 of T. b. ssp. and T. cruzi highlighted a selective toxicity toward the latter, which is a prerequisite for a potential new drug. It may be noted that the activity encountered with the crude CH2Cl2 extract on T. b. brucei (STIB 427 strain) cannot be attributed entirely to the isolated pure compounds. Cytotoxicity was observed against mouse skeletal L-6 cells, and the selectivity indices were lower than the reference value of 50 required to be considered as a hit,22 except for antidesmone 10. The latter was patented in 2003 for its potential as an antiprotozoal drug.20 The good activity and selectivity toward T. cruzi encourage further investigations to elucidate the mechanism(s) of action and to look for similar structures with reduced toxicity as possible antichagasic drugs.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation was measured on a JASCO P-1030 (Easton, MD, USA) polarimeter (EtOH, c in g/100 mL). UV spectra were recorded on a PerkinElmer Lambda-25 UV−vis spectrophotometer (Wellesley, MA, USA) in MeOH. IR spectra were measured on a PerkinElmer Spectrum 100. 1 H and 13C NMR spectra were recorded on a Varian Unity Inova 500 MHz NMR (Palo Alto, CA, USA) instrument. Chemical shifts are reported in parts per million (δ) using the residual CD3OD signal (δH 3.31; δC 49.0) or the CDCl3 signal (δH 7.26; δC 77.2) as internal standards for 1H and 13C NMR, respectively, and coupling constants (J) are reported in Hz. HRMS spectra were obtained on a Q Exactive Plus Hybrid quadripole-orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) using electrospray in the positive mode. The spray voltage was set at 3.5 kV; the sheath gas flow rate (N2), 50 units; the capillary temperature, 320 °C; the S lens RF level, 50; and the probe heater temperature, 425 °C. UHPLC was performed on an Ultimate 3000 UPLC System (Thermo Scientific) with a Kinetex core shell C18 column (2.6 μm; 100 × 3.0 mm i.d.; Phenomenex). Medium-pressure liquid chromatography was performed using a Shimadzu LC10AD pump equipped with a Knauer UV detector and an MPLC glass column (460 × 70 mm i.d.) loaded with ZEOprep C18 as the stationary phase (15−25 μm, Zeochem AG, Uetikon am See, Switzerland). Semipreparative chromatography was performed on an ARMEN Spot System (Saint-Avé, France) with a Kinetex Axia Core−Shell C18 column (5 μm, 250 × 21.2 mm; Phenomenex). ECD Computational Details. Conformational analysis was performed with MacroModel 9.1 software (Schrödinger LLC, New York) using the OPLS 2005 (Optimized Potential for Liquid Simulations) force field in H2O. Conformers occurring within a 2 kcal/mol energy window from the global minimum were chosen for geometrical optimization and energy calculation using density function theory (DFT) with the B3LYP functional and the 6-31G** basis set in the gas phase with the Gaussian 09 program.23 Vibrational analysis was done at the same level to confirm minima. TD-DFT/B3LYP/6-31G** was conducted in MeOH using the SCRF method, with the CPCM model. ECD curves were constructed on the basis of rotatory strength dipole velocity (Rvel), and dipole lengths (Rlen) were calculated with a half-band of 0.2 eV using SpecDis v1.61.24 Plant Material. The roots of Waltheria indica were collected between June 2012 and February 2013 in Zinder (Niger). Their identification was confirmed by Didier Roguet (Botanical Garden of Geneva). Voucher specimens are deposited at the Botanical Garden of Geneva (no. 19003). F

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and 3; HRESIMS m/z 366.2640 [M + H]+ (calcd for C21H36NO4, 366.2644). Waltherione L (9): pale yellow oil; UV (MeOH) λmax (log ε) 207 (5.4), 264 (1.1), 331 (0.7) nm; ECD (MeOH, c 0.13 mM, 0.1 cm) [θ]208 = +63 380, [θ]222 = −54 802, [θ]233 = +13 370, [θ]256 = +12 098; IR (CHCl3) νmax 2931, 2855, 1585, 1486, 1456, 1276 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 370.2375 [M + H]+ (calcd for C23H32NO3, 370.2382). Antidesmone (10): pale yellow oil; HRESIMS m/z 320.2222 [M + H]+ (calcd for C19H30NO3, 320.2226); for other spectroscopic data, see Bringmann et al.18 Antitrypanosomal and Cytotoxicity Assays. The in vitro activities against T. b. rhodesiense and T. cruzi as well as cytotoxicity assessment in L6 cells were determined as reported elsewhere.25 The following strains and parasite forms were used: T. b. rhodesiense, STIB900, trypomastigote forms; T. cruzi, Tuluhaen C2C4 (LacZ), amastigotes and L6 cells, rat skeletal myoblasts. Results are expressed in μg/mL for plant extracts and in μM for pure compounds. Trypanosoma brucei brucei Assay. The New York single marker (NYSM) cell line of bloodstream form of T. b. brucei was cultured in HMI-9 medium supplemented with 10% FCS and used as a wild type. IC50 values were measured using the AlamarBlue assay26 in a 96-well plate in a final volume of 100 μL and a final cell concentration of 0.5 × 105 cells/mL. After 46 h of incubation with the compounds at different dilutions, the cells were incubated two more hours with the AlamarBlue reagent. The fluorescence was detected by excitation at 530 nm and recording the emission at 584 nm with a fluorescence scanner (SynergyMx, Biotek). IC50 values were calculated by nonlinear fitting to the sigmoidal dose−response curve using Prism 6.0 (GraphPad Software, San Diego, CA, USA). The assays were performed in triplicates.

Extraction and Isolation. Air-dried roots (3.0 kg) were powdered and extracted with 3 L of CH2Cl2 (3 × 24 h) at room temperature. After filtration, the CH2Cl2 extract was evaporated to dryness. The residue (2.3 g) was mixed with 8 g of the ZEOprep C18 stationary phase and introduced into an MPLC column by dry injection. Fractionation was carried out with a 460 × 70 mm i.d. column, ZEOprep C18, 15−25 μm, as the stationary phase with MeOH and H2O containing 0.1% formic acid in an optimized gradient mode: 10% to 60% (in 31 h), a plateau at 60% (for 30 h), and finally 60% to 100% (in 24 h) MeOH. The flow rate was set to 6 mL/min, and the UV detection was performed at 220 nm. The MPLC separation yielded 320 fractions, which were individually analyzed by UHPLC-MS and gathered in 90 fractions. Fraction 49 yielded 1 (12.7 mg), fraction 61 yielded 2 (36.9 mg), and fraction 76 yielded 3 (9.3 mg). Fractions 65, 66, 69, 73, and 79 were selected for further purification. The final fractionation steps were performed by semipreparative HPLC using a Kinetex Axia Core−Shell C18 column (5 μm, 250 × 21.2 mm; Phenomenex) using MeCN/H2O/0.1% formic acid as solvents for the isocratic elution. The flow rate was set to 25 mL/min, and UV absorbance was at 220 nm. Fraction 65 (166.0 mg) yielded 4 (84.0 mg) and 10 (10.1 mg) (46% MeCN), fraction 66 (30.3 mg) yielded 4 (6.8 mg) and 5 (1.6 mg) (46% MeCN), fraction 69 (10.0 mg) yielded 6 (1.5 mg) (46% MeCN), fraction 73 (30.0 mg) yielded 7 (3.2 mg) (51% MeCN), and fraction 79 (10.7 mg) yielded 9 (1.7 mg) (55% MeCN). 8-Deoxoantidesmone (1): pale yellow oil; [α]22 D −10 (c 0.05, EtOH); UV (MeOH) λmax (log ε) 219 (3.4), 272 (2.0) nm; ECD (MeOH, c 0.16 mM, 0.1 cm) [θ]261 = −27 489, [θ]285 = +10 237; IR (CHCl3) νmax 2924, 2851, 1617, 1500 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 306.2428 [M + H]+ (calcd for C19H32NO2, 306.2433). Waltherione E (2): pale yellow oil; [α]22 D +61 (c 0.1, EtOH); UV (MeOH) λmax (log ε) 220 (4.6), 273 (2.8) nm; ECD (MeOH, c 0.17 mM, 0.1 cm) [θ]226 = +214 875, [θ]265 = +309 309, [θ]285 = −141 518; IR (CHCl3) νmax 2923, 2853, 1617, 1504, 1268, 1228, 1099 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 336.2534 [M + H]+ (calcd for C20H34NO3, 336.2539). Waltherione F (3): pale yellow oil; [α]22 D +2 (c 0.05, EtOH); UV (MeOH) λmax (log ε) 235 (2.3), 332 (7.4) nm; IR (CHCl3) νmax 3261, 2925, 2854, 1714, 1625, 1576, 1529, 1459, 1259, 1237 cm−1; 1H and 13 C NMR, see Tables 2 and 3; HRESIMS m/z 332.2220 [M + H]+ (calcd for C20H30NO3, 332.2226). Waltherione G (4): pale yellow oil; 2: colorless oil; [α]22 D +33 (c 0.1, EtOH); UV (MeOH) λmax (log ε) 208 (5.4), 280 (3.1) nm; ECD (MeOH, c 0.15 mM, 0.1 cm) [θ]232 = +95 660, [θ]265 = +154 243, [θ]295 = 56 323; IR (CHCl3) νmax 2930, 2855, 1615, 1563, 1452, 1276, 1128 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 370.2378 [M + H]+ (calcd for C23H32NO3, 370.2382). Waltherione H (5): pale yellow oil; UV (MeOH) λmax (log ε) 205 (4.7), 284 (3.2) nm; ECD (MeOH, c 0.15 mM, 0.1 cm) [θ]231 = +85 561, [θ]273 = +125 543, [θ]300 = −52 933; IR (CHCl3) νmax 2929, 2855, 1603, 1569, 1453, 1276 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 400.2483 [M + H]+ (calcd for C24H34NO4, 400.2488). Waltherione I (6): pale yellow oil; UV (MeOH) λmax (log ε) 205 (5.3), 223 (2.2), 284 (1.3) nm; ECD (MeOH, c 0.14 mM, 0.1 cm) [θ]271 = −133 366, [θ]300 = +39 348; IR (CHCl3) νmax 3257, 2925, 2855, 1601, 1547, 1457, 1277 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 352.24831 [M + H]+ (calcd for C20H34NO4 352.24879). Waltherione J (7): pale yellow oil; UV (MeOH) λmax (log ε) 207 (5.7), 221 (2.5), 281 (1.5) nm; ECD (MeOH, c 0.15 mM, 0.1 cm) [θ]233 = +64 370, [θ]268 = +116 139, [θ]300 = −44 876; IR (CHCl3) νmax 2923, 2853, 1607, 1564, 1458, 1276 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 336.2534 [M + H]+ (calcd for C20H34NO3, 336.2539). Waltherione K (8): pale yellow oil; UV (MeOH) λmax (log ε) 208 (5.7), 284 (1.4) nm; ECD (MeOH, c 0.14 mM, 0.1 cm) [θ]232 = +2 448 934, [θ]268 = +364 776, [θ]300 = −156 693; IR (CHCl3) νmax 2924, 2855, 1605, 1570, 1453, 1276 cm−1; 1H and 13C NMR, see Tables 2

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of compounds 1 to 9 are available. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +41 22 379 65 61. Fax: +41 22 379 33 99. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank M. Cal, S. Keller, and M. Jud (Swiss TPH) for parasite assay results. REFERENCES

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